Exogenous AMPA downregulates gamma-frequency network oscillation in CA3 of rat hippocampal slices

Pharmacologically-induced persistent hippocampal γ oscillation in area CA3 requires activation of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs). However, we demonstrated that exogenous AMPA dose-dependently inhibited carbachol (CCH)-induced γ oscillation in the CA3 area of rat hippocampal slices, but the underlying mechanism is not clear. Application of AMPARs antagonist NBQX (1 μM) did not affect γ oscillation power (γ power), nor AMPA-mediated γ power reduction. At 3 μM, NBQX had no effect on γ power but largely blocked AMPA-mediated γ power reduction. Ca2+-permeable AMPA receptor (CP-AMPAR) antagonist IEM1460 or CaMKK inhibitor STO-609 but not CaMKIIα inhibitor KN93 enhanced γ power, indicating that activation of CP-AMPAR or CaMKK negatively modulated CCH-induced γ oscillation. Either CP-AMPAR antagonist or CaMKK inhibitor alone did not affected AMPA-mediated γ power reduction, but co-administration of IEM1460 and NBQX (1 μM) largely prevented AMPA-mediated downregulation of γ suggesting that CP-AMPARs and CI-AMPARs are involved in AMPA downregulation of γ oscillation. The recurrent excitation recorded at CA3 stratum pyramidale was significantly reduced by AMPA application. Our results indicate that AMPA downregulation of γ oscillation may be related to the reduced recurrent excitation within CA3 local neuronal network due to rapid CI-AMPAR and CP-AMPAR activation.

At a concentration of 3 μM, NBQX had no effect on CCH-induced γ oscillation, but affected the dynamics of AMPA modulation of γ oscillation. In the presence of NBQX, AMPA(1 μM) no longer caused a constant suppression of γ oscillations, but sometimes an initial temporary downregulation followed by a rapid recovery of γ power or little inhibition on γ oscillations (99 ± 1% of NBQX, vs NBQX or CCH, P > 0.05, n = 6 slices from 3 rats, Fig. 2F-J). These results indicate that AMPA-mediated downregulation of γ oscillation was involved in CI-AMPAR activation.
The effects of CAMKK inhibitor STO-609 and CAMKII inhibitor KN93 on AMPA-mediated reduction of γ oscillation. The inhibitory neurons express relatively low NMDAR but abundant CP-AMPAR, which is a main source for the dendritic Ca 2+ signaling of interneurons 36 . CP-AMPAR-mediated Ca 2+ influx may trigger endoplasmic Ca 2+ release from ryanodine receptor, which amplify Ca 2+ signaling and mediate synaptic transmission and plasticity in interneurons 37,38 . Previous study also showed that CP-AMPARs contribute to activity-dependent CaMKII T286 phosphorylation 39 . It is thus possible that activation of CP-AMPAR may affect CaMKK/CaMKII signaling.
The effect of AMPA on the synaptic transmission of CA3 from mossy fibers and CA3 recurrent collateral fibers. Previous study demonstrated that perfusion of exogenous NMDA to the hippocampal slice depressed glutamatergic transmission 40 . Consistent with this idea, it is critical to demonstrate the effect of AMPA on the synaptic transmission of area CA3 (mossy fibers and recurrent collateral fibers). It is likely that AMPA-mediated reduction in the oscillatory activity may be due to a depression of glutamatergic transmission in CA3 area. To test this hypothesis, we then recorded the mossy fibers-CA3 synaptic transmission by placing www.nature.com/scientificreports/ a stimulus electrode in mossy fibers from dentate gyrus (see Fig. 6A) and a record electrode in CA3 stratum pyramidale, and we found that AMPA (5 μM) had no effect on the slope of field excitatory synaptic potential (fEPSP) evoked by mossy fiber stimulation (102.7 ± 8% of Ctrl, vs Ctrl, t (26) = 0.403, P = 0.69, n = 14 slices from 6 rats for both Ctrl and AMPA group, Fig. 6A-D). We next recorded the recurrent excitation by placing a stimulus electrode in CA3 stratum radiatum and recording electrode in CA3 pyramidal lawyer and found that AMPA (5 μM) significantly reduced the slope of fEPSP (15.63 ± 4.1% of Ctrl, vs Ctrl, t (20) = 29.132, P < 0.001, n = 11 slices from 5 rats for both Ctrl and AMPA group, Fig. 6A,E-G). These data indicate that AMPA downregulation of γ power may be associated with the reduced synaptic transmission of CA3 from recurrent collateral fibers.

Discussion
AMPARs play a critical role in fast excitatory synaptic transmission and synaptic plasticity 41,42 . Numerous evidence showed multiple neurological disorders are involved in dysregulation of plasticity processes or excitatory synaptic transmission 43 . γ oscillations are generated by interaction between excitatory and inhibitory neurons 44 . In this study, we demonstrated that exogenous AMPA inhibited γ power and increased the peak frequency of CCH-induced γ oscillations in the CA3 area of the rat hippocampal slices. AMPA-mediated downregulation of γ power and www.nature.com/scientificreports/ increase in peak frequency suggests that neurons were able to oscillate at higher frequency given that these neurons no longer need to support oscillation at a higher power. The frequency of CCH-induced γ oscillations in the mouse hippocampal CA3 was increased by the activation of NMDA receptors (NMDARs) on interneurons 45 . Since NMDAR antagonist had no effect on AMPA mediated γ, it is currently difficult to link a role of NMDAR in AMPA-induced increase in peak frequency.
CCH-induced γ oscillations in the hippocampal CA3 depend on AMPAR-mediated excitation 45 . Hippocampal CA3 area enriched in recurrent collateral which are critical for generation of γ oscillations in CA3. Contrary to the common belief, perfusion of NMDA to the hippocampal slices depressed glutamatergic transmission 40 . In this study, we demonstrated the significant inhibitory effect of exogenous AMPA on the synaptic transmission of area CA3 from recurrent collateral fibers but not from mossy fibers. Thus, the reduction in the oscillatory activity may be the consequence of AMPA-mediated depression of glutamatergic transmission similar to that observed with NMDA in area CA1 of the hippocampus 40 .
Ca 2+ participates in multiple important physiological activities including network oscillation and synaptic plasticity 46 . We previously demonstrated that L-type calcium channel activation correlated with rapid γ power reduction 47 . In this study, nifedipine had no effect on AMPA downregulation of γ power, suggesting L-type calcium channel was not involved in the role of AMPA.
Either a low concentration of NBQX (1 μM) or IEM1460 had no effect on AMPA-mediated γ reduction, whereas co-administration of IEM1460 and NBQX largely blocked AMPA-mediated γ reduction, indicating the potentiated effect of the combined antagonists on CP-AMPAR and/or CI-AMPAR.
Recently, Pampaloni et al. 49 identified the slow AMPAR in the hippocampus which provides massive depolarization that can trigger an action potential from a single stimulation and produce short-term potentiation from a purely postsynaptic locus. Whether the slow AMPAR is involved in hippocampal γ oscillations remains to be further determined. Currently we cannot exclude the possibility of slow AMPAR involvement in AMPAmediated downregulation of γ oscillations.
The rapid downregulation of AMPA on γ oscillation found in this study provides a tool for the rapid, reversible modulation of γ oscillations.γ oscillations are believed to exert a role in cognition and aberrant γ oscillations observed in AD 12 . Studies in APP/PS1 AD mice have shown that prior to significant neuropathological changes, www.nature.com/scientificreports/ the expression and phosphorylation of CP-AMPAR were increased, which was involved in the regulation of synaptic plasticity at early stages of AD 50 . AD-associated increase in the expressions of CP-AMPAR also linked to abnormal neural network activity 51 . CP-AMPAR inhibition may serve as a strategy for the intervention of neurodegenerative diseases in which calcium homeostasis was disturbed and γ oscillation was impaired. In this study, we demonstrated that CP-AMPAR antagonist IEM1460 and CaMKK inhibitor STO 609 significantly enhanced γ oscillations, IEM1460 and STO 609 alone or in combination may therefore serve as one potentially therapeutic strategy for the AD treatment. Our study provided not only novel insights into the exogenous AMPAmediated hippocampal γ oscillation but also a strategy for boosting hippocampal γ oscillation, which is critical for the improvement of cognitive function in many neurological disorders. www.nature.com/scientificreports/ experimental protocol (protocol code: XYLL20210120, date of approval: March 8, 2021). Utmost efforts were made to decrease animal suffering as well as the number of animals utilized in current study. Prior to the operation, intraperitoneal injection of Sagatal (sodium pentobarbitone, 100 mg kg-1, Rhône Mérieux Ltd., Harlow, United Kingdom) was performed to anesthetize the animals. By the time all pedal reflexes disappeared, the rats were perfused intracardially with oxygenated saturated chilled (5 °C) artificial cerebrospinal fluid (ACSF) in which sodium chloride was replaced with iso-osmotic sucrose. This sucrose-ACSF contained (in mM): 3 KCl, 225 sucrose, 1.25 NaH 2 PO 4 , 24 NaHCO 3 , 0.5 CaCl 2 , 6 MgSO 4 , and 10 glucose (pH: 7.4). The rat brains were then undergone horizontally cutting (400 μm) at 5 °C in the sucrose-ACSF with a Leica VT1000S vibratome (Leica Microsystems, Milton Keynes, United Kingdom). The brain slices were stored in a chamber containing sucrose-ACSF prior to extracellular field recordings.
Electrophysiological recording, data acquisition and analysis. The brain slices were allowed to equilibrate for 1 h in the ACSF saturated with humidified carbogen (95% O 2 and 5% CO 2 ) at 32 °C in the Haas type chamber before recording. Glass microelectrodes containing ACSF (resistance 2-5 MΩ) were employed to record the extracellular field potentials at CA3c stratum pyramidale of rat hippocampus. A NL100AK AC pre-amplifier headstage was used to increase the signal-to-noise ratios before the pulses enter an amplifier (Digitimer Ltd., Welwyn Garden City, United Kingdom). A Neurolog NL106 AC/DC amplifier (Digitimer) was used to amplify field potential and a Neurolog NL125 filter (Digitimer) was utilized for band-pass filter between 0.5 Hz and 2 kHz. The electromagnetic interference originating from the main power supply was eliminated from the extracellular field recordings with one 50 Hz Humbug noise eliminator (Digitimer Ltd.). The extracellular field recording signals were digitized at a sample rate of 5-10 kHz using a CED 1401 plus ADC board (Cambridge Electronic Design, Cambridge, United Kingdom).
The recorded data were analyzed offline with Spike2 software (Cambridge Electronic Design). To provide a quantitative measure of frequency components, power spectra were also plotted using Spike2 software. The power spectra were constructed for 60 s epochs with a fast Fourier transform algorithm. The area under the curve between 20 and 60 Hz was used to quantify the γ power. Autocorrelograms were calculated with Spike2 software using a 500-ms lag from the same local field potential recording employed for γ power calculation. It is generated by fitting the autocorrelation peaks with an exponential function(Y = exp(− Δt/τ)). The decay time constant (τ) of the autocorrelation peaks is utilized to measure the regularity of the oscillation.
Statistical methods. All statistical tests were performed using SigmaStat software (Sysstat software, San Jose, CA, United States). Where data sets were not different from a normal distribution, results are expressed as mean ± standard error of the mean, between-group comparisons were made with a Student's t-test and withingroup comparisons were made with a paired Student's t-test or a repeated measures ANOVA. Where data sets were different from a normal distribution, results are expressed as median (interquartile range) and betweengroup differences were tested with a Mann-Whitney U tests. Effects were considered statistically significant if P < 0.05. *, **, *** represents P < 0.05, 0.01, 0.001, respectively.
Drugs. CCH was purchased from Sigma. The selective AMPAR agonist AMPA, AMPAR antagonist NBQX, CP-AMPAR antagonist IEM1460, CAMKK inhibitor STO-609, CAMKII inhibitor KN93 and L-type calcium channel blocker nifidepine were purchased from Tocris Bioscience (Bristol, UK). Stock solutions such as AMPA, NBQX, nifidepine, STO-609, KN93, CCH and IEM1460, at thousand times the final concentration, were dissolved in DMSO, and stored in individual aliquots at − 20 °C. Final solutions were prepared freshly on the day of the experiment.

Data availability
Data will be available from the corresponding author on request.